(PART A) REGENERATION OF ZEA MAYS PLANTS FROM PROTOPLASTS
Descriptions of regeneration of Zea mays from callus mainly date from the mid 1970's" [for example: Green and Phillips, Crop Sc., 15 (1975) 417-421,; Harms et al. Z. Pflanzenzuechtg., 77 (1976) 347-351; European patent applications EP-0,160,390, Lowe and Smith (1985); EP-0,176,162, Cheng (1985); and EP-0,177,738, Close (1985)]. Attempts have been made to obtain division and subsequent plant regeneration from protoplasts of Zea mays over a period of more than 12 years [Potrykus et al., In: Cell Genetics in Higher Plants, Dudits et al., (eds), Akademiai Kiado, Budapest (1976) 129-140, and references therein; Harms, "Maize and Cereal Protoplasts-Facts and Perspectives," Maize for Biological Research, W. F. Sheridan, ed. (1982); Dale, in: Protoplasts (1983); Potrykus et al (eds.) Lecture Proceedings, Experientia Supplementum 46, Potrykus et al., eds, Birkhauser, Basel (1983) 31-41, and references therein]. However, protoplasts of Zea mays have always given rise only to cultures which have not been capable of regenerating fertile plants [Potrykus et al., Mol. Gen. Genet. 156 (1977) 347-350; Potrykus et al., Theor. Appl. Genet., 54 (1979) 209-214; Choury and Zurawski, Theor. Appl. Genet., 59 (1981) 341-344; Vasil, In: Proc. 5th Int. Cong. Plant Tissue and Cell Culture, Fujiwara (ed), Tokyo, (1982) 101-104; Imbrie-Milligan and Hodges, Planta, 168, (1986) 395-401; Vasil and Vasil, Theor. Appl. Genet., 73 (1987) 793-798]. However, none of these references provides a description of a procedure for producing protoplasts capable of regenerating fertile plants have appeared.
The ability to produce such protoplasts would allow the genetic manipulation of this important crop by, for example, transformation or fusion, or the use of protoplasts as the starting material for selection of valuable new phenotypes.
Although there has been great interest in genetic transformation of Zea mays, there has been no description to date of a successful in vitro method which can lead to a regenerated, transformed plant, although there have been descriptions of transformation of Zea mays protoplasts that have led to the recovery of non-regenerable callus [Fromm et al., Nature, 319, (1986) 791-793]. One method to produce transgenic Zea mays plants would be to find a way in which to stably transform protoplasts capable of regeneration to whole fertile plants. However no description of a method to produce Zea mays protoplasts capable of undergoing differentiation to whole fertile plants has appeared.
This and other objectives have been achieved in accordance with the present invention by discovering a method for producing Zea mays protoplasts that can divide to form callus colonies. The protoplasts can be transformed, and the calli are capable of regenerating fertile Zea mays plants. The process for producing Zea mays protoplasts capable of dividing and forming callus, which can then be induced to regenerate into whole fertile plants requires a special type of callus. Such callus and methods for producing and identifying it will be described, and are considered part of the invention.
The special type of callus is used to initiate cell suspensions, which are also considered part of the invention. These cell suspensions, upon subculture, give rise to suspensions which can be used to isolate protoplasts. These protoplasts are capable of dividing and forming callus, which can then be induced to regenerate plants. Cell division or callus formation is promoted by the presence of certain agents, such as O-loweralkanoyl-salicylic acid (O-acetyl-salicylic acid and its homologs), plant growth regulators or DMSO, or by combinations of these agents.
Plant regeneration from protoplast-derived Zea mays cells in culture is essential for the application of somatic hybridization, for the production of improved Zea mays varieties through somaclonal variation, and for the use of genetic engineering in producing new Zea mays plants, plant varieties, cultivars or inbreds with new phenotypic traits.